Noncanonical α-amino acids (ncAAs) resemble the building blocks of natural proteins but are not themselves used in protein synthesis. Despite this, ncAAs are prevalent precursors for functional synthetic compounds, including over 12% of the 200 top-selling pharmaceuticals.1 However, ncAAs are challenging synthetic targets, since they possess at minimum two reactive functional groups (the amine and carboxylic acid) and typically have at least one stereocenter. As a result, synthetic routes to ncAAs typically require multiple steps, most of which use organic solvents.2,3 One of the most direct routes to ncAAs is to add a nucleophile to the β-position of a serine-derived lactone4-6 or aziridine7-8(FIG. 1A), but this approach has certain drawbacks, such as the need to pre-synthesize the water-sensitive electrophilic reactants.
Enzymes are widely applied to the synthesis of ncAAs since they circumvent many of the limitations of chemical methods. Not only do these catalysts function in aqueous media, but they also exhibit chemoselectivity that obviates the need for protecting groups, thereby trimming synthetic steps. In addition, the reactions are often highly stereoselective. Unfortunately, most enzymatic methods to synthesize ncAAs, such as those that rely on hydrolases or transaminases, require that the majority of the final product be synthesized in advance, usually by chemical means, with the enzyme only appearing at the end to set the stereochemistry. By contrast, enzymes like tryptophan synthase,9-14 which uses the cofactor pyridoxal 5′-phosphate (PLP, FIG. 1B), can form ncAAs by nucleophilic substitution at the β-position of readily available amino acids like serine. In this reaction scheme, the enzyme forms an active electrophilic species, the amino-acrylate (FIG. 1C), directly in the active site, which is then intercepted by a nucleophilic substrate. These reactions can be run in aqueous conditions that would hydrolyze the serine-derived lactones or aziridines. Furthermore, the enzyme active site can bind the substrates to accelerate the reaction and control the regioselectivity of nucleophilic substitution.
The ncAA 4-cyanotryptophan (4-CN-Trp) was previously reported to exhibit blue fluorescence (Amax=405 nm) with a high quantum yield and long lifetime.15 These properties, among others, make 4-CN-Trp an attractive small-fmolecule fluorophore for imaging studies in vitro and in vivo. However, the chemical synthesis requires multiple steps, including a low-yielding Pd-catalyzed cyanation reaction.